Programmable aptazymes integrated with fluorescence light-up aptamer reporters enable universal, label-free detection of diverse molecular targets

Aptazymes, which integrate molecular recognition with catalytic activity, are promising signal transducers in biosensing applications. However, their utility is constrained by limited modularity and the reliance on costly signal-labeling strategies. Here, we present a programmable aptazyme platform incorporating a shared catalytic module that enables a generalized, label-free fluorescence detection of diverse molecular targets. Each aptazyme comprised a conserved flanking arm, catalytic core, and linker, with a target-specific aptamer domain. Target binding allosterically activated the catalytic function, triggering substrate cleavage and the release of a uniform single-stranded DNA (ssDNA) output, irrespective of the input molecule. A fluorescence light-up aptamer reporter, assembled from two probe strands, recognized the released ssDNA to form a tripartite complex that activated fluorescence in a label-free manner. Using ATP, quinine, and phenylalanine as model targets, the platform demonstrated robust sensing performance with a linear range of 5–150, 5–200, and 20–500 μM, respectively, and a limit of detection (LOD) of 1.03, 1.51, and 1.68 μM, respectively. This all-DNA biosensing system is cost-effective and modular for detecting various analytes. We anticipate that this strategy can be broadly adapted by incorporating alternative aptamers and DNAzymes.